John Wallace

Microbial ecology of the rumen

The rumen

The three domains of life

Ruminal microorganisms

50 m

0.5 m

100 m

Ciliate protozoa

106 per g digesta

Anaerobic fungi

103 per g digesta

50 m

Bacteria

1010 per g digesta

Methanogenic archaea

108 per g digesta

2 m

Rumen ciliate protozoa

100 m

Enchelyodon sp.

Amylovorax dehorityi Bitricha tasmaniensis

Amylovorax dogieli Dasytricha ruminantium U57769 Dasytricha ruminantium (France)

Dasytricha ruminantium U27814 Epidinium caudatum

Ophryoscolex purkynjei RS65

Polyplastron multivesiculatum U27815 Polyplastron multivesiculatum (Poland) RS53

Polyplastron multivesiculatum U57767 Polyplastron multivesiculatum (France)

Diplodinium dentatum Ostracodinium dentatum (Poland)

Enoploplastron triloricatum (Slovakia) Eudiplodinium maggii (France) Eudiplodinium maggii (Poland) Eudiplodinium maggii RS61, RS99

Diploplastron affine (Poland) RS59

RS70 RS88, RS33

RS7 RS87

RS24 RS74

RS17 RS100, RS95

RS28 RS2

RS75 RS31

RS57 RS86

RS90 RS94

RS67, RS1 RS58, RS18, RS16, RS3

Entodinium nanellum (Slovakia) RS14 RS89, RS97, RS63, RS13

RS105 Entodinium furca monolobum (Slovakia)

RS26 RS30

RS85 RS77

RS73 RS12 RS4

RS79 Entodinium bursa (Slovakia)

RS71 RS64

Entodinium caudatum

Entodinium caudatum (UK) RS62

RS82, RS32, RS69, RS66, RS22 RS9

Entodinium caudatum (Slovakia) RS92

RS93 Entodinium D?( France)

RS5, RS107 Isotricha intestinalis Isotricha intestinalis (Poland)

Isotricha prostoma (Slovskia) Isotricha prostoma (Poland) Isotricha prostoma (France)

RS19

100

100

73

97 100

100 91 92

100

100 96

94

94

100

77

95

85

69

79

Didinium nasutum

Unexpected protozoal diversity

Wright et al (1997). J Eukar

Microbiol 44, 61-67

Fungal picture

Rumen anaerobic fungi

50 m

Rowett picture

0.5 m

Rumen bacteria

ProteobacteriaM ethanomicrobium mobile (M59142)

0.1

82.7

88.3

Proteobacter ia (OTUs 1-3)

Cytophaga-Flexibacter-Bactero ides Group (OTUs 6-78)

Low G+C Gram Positive Bacteria (OTUs 80-174)

Fibrobacter Group (OTU 175)High G+C Gram Positive Bacteria (OTUs 176 &177)

Chlamydiales -Verrucimicrobia Group (OTUs 1 78 & 179)Spirochaetes (180)

Phylum? (OT U 4)

Phylum? (OTU 5)

Phylum? (OTU 79)

Figure 2

Figure 1: Phylogenetic tree of 16S rDNA rumen library sequence data.

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Bacteroidetes

Proteobacteria

Firmicutes

Fibrobacteraceae

Spirochaetes

Edwards et al. (2004) Anton v

Leeuwen 86, 263-281

Rumen methanogenic archaea

2 m

Phylogenetic dendrogram of total rumen and rumen protozoan-associated archaea and

selected reference sequences.

Janssen P H , and Kirs M Appl. Environ. Microbiol.

2008;74:3619-3625

Methanobrevibacter

usually predominant

genus (60-99%)

Phylogenetic dendrogram of total rumen and rumen protozoan-associated archaea and

selected reference sequences.

Janssen P H , and Kirs M Appl. Environ. Microbiol.

2008;74:3619-3625

‘Rumen Cluster C’

Methanoplasmata

Probably methylotrophic

(e.g. methylamine CH3-

NH2)

Methane production in the rumen

Fermentation

H2 + CO2

CH4

Protozoa, fungi, bacteria

Archaea

Methane production in ruminants

Respiration chambers SRUC, Edinburgh

• 36 Aberdeen Angus, 36 Limousin cross steers, 1-2 years

• Two diets: Forage diet 50:50, forage (grass silage / whole crop silage) : concentrate (barley / barley dark grains ) (n=36) Concentrate diet 8:92, forage (straw) : concentrate (barley / barley dark gains) (n=36)

• Methane emissions measured for 2 days in Green Cow facility; ruminal digesta taken by stomach tube

• Animals slaughtered 1-3 weeks later; ruminal digesta taken again

• Archaea and bacterial 16S rRNA genes measured by qPCR

• Data analysis, GLM procedure of SAS

The Extreme 8 experiment

Selected the 4 highest and 4 lowest methane

emitters for full microbiome/metagenome analysis

Microbiome

The Extreme 8 analysis

Microbiota

Metagenome

What species are

different?

What genes are

different?

Secondary structure

of small

subunit ribosomal

RNA

The Extreme 8 microbiota analysis

Variation in methane production: microbiota analysis

Methane

0

5

10

15

20

25

30

LOW HIGH

Met

han

e (g

/kg

DM

I)

P = 0.004

0

0,5

1

1,5

2

2,5

3

3,5

4

LOW HIGH

Ab

un

da

nce

(%

)

Archaea

0

2

4

6

8

10

12

LOW HIGH

Ab

un

dan

ce (

%)

Succinovibrionaceae

P = 0.014P = 0.002

Methane emission status of beef cattleFour highest and four lowest from 72 beef cattle

Herbivore

Foregut fermenter

Produces 20% of methane per DMI

compared to cattle

Herbivore

Foregut fermenter

Has large numbers of

Succinovibrionaceae

Microbiome

The Extreme 8 analysis

Microbiota

Metagenome

What species are

different?

What genes are

different?

Variation in methane production: the geneticist’s slant

KEGG ID/Diet Description bEstimate cVIP

K06001 Tryptophan synthase beta chain [EC:4.2.1.20] 0.13310 1.233

Diet conc Concentrate based diet -0.14695 1.204

Diet mixed Mixed forage-concentrate diet 0.14695 1.204

K02118 V/A-type H+-transporting ATPase subunit B [EC:3.6.3.14] 0.07984 1.151

K02117 V/A-type H+-transporting ATPase subunit A [EC:3.6.3.14] 0.08564 1.133

K00638 Chloramphenicol O-acetyltransferase [EC:2.3.1.28] 0.06182 1.082

K00200 Formylmethanofuran dehydrogenase subunit A [EC:1.2.99.5] 0.06042 1.070

K03531 Fell division protein FtsZ 0.07526 1.065

K00201 Formylmethanofuran dehydrogenase subunit B [EC:1.2.99.5] 0.04859 1.049

K00399 Methyl-coenzyme M reductase alpha subunit [EC:2.8.4.1] 0.03843 1.021

K00123 Formate dehydrogenase, alpha subunit [EC:1.2.1.2] 0.03417 1.013

K03388 Heterodisulfide reductase subunit A [EC:1.8.98.1] 0.02734 0.997

K14126 F420-non-reducing hydrogenase subunit A [EC:1.12.99.-] 0.00384 0.933

K01079 Phosphoserine phosphatase [EC:3.1.3.3] 0.03424 0.932

K00401 Methyl-coenzyme M reductase beta subunit [EC:2.8.4.1] 0.00484 0.909

K00527 Ribonucleoside-triphosphate reductase [EC:1.17.4.2] 0.03449 0.898

K02337 DNA polymerase III subunit alpha [EC:2.7.7.7] -0.05338 0.886

K02837 Peptide chain release factor RF-3 0.00883 0.852

K01893 Asparaginyl-tRNA synthetase [EC:6.1.1.22] -0.06673 0.812

K00925 Acetate kinase [EC:2.7.2.1] -0.00903 0.812

K00656 Formate C-acetyltransferase [EC:2.3.1.54] -0.00834 0.787

K02948 Small subunit ribosomal protein S11 -0.00400 0.734

Rainer Roehe

SRUC

“The genes identified in Table 3 to be important

explained the variation in methane emissions by 88%.”

Ru

min

Om

ics

Tools, resources, legacy

• Proxies Buccal-ruminal-faecal microbiomes

Proxies for rumen sampling – mouth (swab and bolus) and faeces

Proxies for rumen sampling – post-mortem samples

y = 1.012xR² = 0.35

0

2

4

6

8

10

12

14

16

18

0 5 10 15

Ch

am

ber

sam

ple

s A

:B r

ati

o

(%)

Post-mortem A:B ratio (%)

Conclusions on methane microbiome research

• Low-methane phenotype has low

archaea and high Succinovibrionaceae

abundance

• Differences in abundance of 22

microbial genes explained the variation

in methane emissions by 88%

• Post-mortem sampling of digesta is a

useful proxy

• Oral samples also a useful proxy

RuminOmics Kevin Shingfield, Ilma Tapio, Johanna Vilkki, Pierre Taberlet, Aurelie Bonin

Methane John Rooke, Rainer Roehe, Mick Watson, Tim Snelling, Graham Horgan

Funders European CommissionBBSRC (UK Research Council)RESAS (Scottish Government)

Acknowledgements